The invention relates to platelet activation proteins.
The normal hemostatic system regulates bleeding and thrombosis through a series of complex interactions between components of the blood vessel wall, circulating blood platelets, and plasma proteins.
Because vascular injury causes a rapid loss of the protein, fluid, and cellular components of the blood, animals have developed rapid responses to patch the vessel and initiate its repair. These rapid responses are initiated by the platelet, a highly specialized cell that reacts to vascular injury. Normally, platelets circulate in the blood as quiescent and nonadherent cells, monitoring the integrity of the blood vessel. In response to vascular injury, platelets adhere to de-endothelialized areas and activate. Platelet activation induces profound morphologic and functional changes in the cell. Platelets change shape, aggregate with other platelets, and adhere to other cells. With full activation, platelets secrete the contents of their lysosomal, alpha, and dense granules, thereby expressing adhesion molecules, growth factors, coagulation enzymes, and other specialized molecules. Molecules expressed by activated platelets execute many of the complex cellular and biochemical processes that staunch the loss of blood and begin the process of vascular repair.
The cellular and biochemical processes initiated by platelets in response to vascular injury can be lifesaving, but in the absence of such injury these same processes can be deleterious. For example, unregulated arterial platelet thrombosis can occlude the blood supply to organs and lead to strokes, heart attacks, and limb necrosis.
A novel polypeptide, designated activated platelet protein-2 (APP-2), which is preferentially expressed on activated human platelets but not resting platelets, has now been discovered.
The invention includes a substantially pure DNA encoding a platelet activation polypeptide having a molecular weight of approximately 25 kilodaltons (kDa). Under non-reducing conditions, it can be naturally found in covalent association with two other proteins in a 145 kDa complex. The 25 kDa polypeptide as expressed in human platelets contains at least 2 putative phosphorylation sites. The protein of the invention can be characterized as containing an epitope which binds to the monoclonal antibody (MAb) 3B2.
Preferably, the encoded polypeptide is human APP-2, which includes at least 95% of the amino acid sequence of SEQ ID NO:4 (e.g. the protein encoded by SEQ ID NO:1). A preferred example of such a DNA would contain the nucleotide sequence of SEQ ID NO:3 or any degenerate variant of SEQ ID NO:3.
Most preferably, the DNA includes the nucleotide sequence of SEQ ID NO:2, or any degenerate variant of SEQ ID NO:2.
A substantially pure DNA containing a strand of at least 12 nucleotides, e.g., a hybridization probe of at least 20 nucleotides, 50 nucleotides, 100 nucleotides or more, which hybridizes at high stringency to a DNA having the sequence of SEQ ID NO:2, or the complement thereof, is also within the invention. Expression of APP-2 in a cell can be detected by (a) contacting mRNA obtained from the cell with a labeled hybridization probe comprising, for example, a single-stranded segment of isolated DNA encoding a fragment of APP-2; and (b) detecting hybridization of the probe with the MRNA.
By xe2x80x9chigh stringencyxe2x80x9d is meant the following DNA hybridization and wash conditions: hybridization at 42xc2x0 C. in the presence of 50% formamide; a first wash at 65xc2x0 C. with 2xc3x97SSC containing 1% SDS; followed by a second wash at 65xc2x0 C. with 0.1xc3x97SSC.
By xe2x80x9csubstantially pure DNAxe2x80x9d is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the DNA sequence of interest. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which (a) is part of a hybrid gene encoding additional polypeptide sequence, e.g., a fusion protein, or (b) has a sequence that is not a naturally-occurring nucleotide sequence (e.g., a degenerate variant of a natural sequence, or a sequence containing mutations which do not occur naturally). Also included is a recombinant DNA which includes a portion of SEQ ID NO:2 and which encodes an alternative splice variant of APP-2, e.g., a polypeptide, the amino terminus of which differs from the amino terminus of SEQ ID NO:1.
The DNA should have at least about 50% identity to the coding sequence of SEQ ID NO:1 or 3, and preferably at least 70% (e.g., 80t, 90% or 95%). The identity between two nucleic acid or polypeptide sequences is a direct function of the number of matching or identical positions. For example, when a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by an adenine, then they are identical at that position. For example, if half, e.g., 5 positions in a sequence 10 nucleotides in length, are identical, then the sequences have 50% sequence identity. The length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). For purposes of calculating % sequence identity, gaps are considered to be mismatches.
The invention also includes a vector containing a DNA encoding a polypeptide which includes the amino acid sequence of SEQ ID NO:1, e.g., a construct in which the coding sequence is operably linked to a promoter or other regulatory sequences for expression of the polypeptide, and a cell containing such a vector. The cell may be procaryotic or eukaryotic (e.g., a mammalian cell such an a human cell) and preferably expresses the recombinant polypeptide encoded by SEQ ID NO:2.
The invention also includes a substantially pure platelet activation polypeptide as described above. By xe2x80x9cplatelet activation polypeptidexe2x80x9d is meant a polypeptide having the amino acid sequence of a protein that is naturally preferentially expressed by activated platelets compared to resting platelets of an animal. Preferably, the animal is a vertebrate, e.g. a mammal such as a primate, including a human; alternatively the mammal is a rat, mouse, rabbit, guinea pig, hamster, cow, pig, horse, goat, sheep, dog, or cat.
Preferably, the polypeptide contains the amino acid sequence of human APP-2 (SEQ ID NO:1), e.g., in the form of a Flag-APP-2 fusion protein. By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). The amino acid sequence of the polypeptide differs solely from SEQ ID NO:1 by conservative amino acid substitutions, e.g., substitution of one amino a acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein (e.g., its binding to Mab 3B2 or its covalent association in the 145 kDa complex). Preferably, the amino acid sequence of the platelet activation polypeptide is at least 50%, more preferably 70%, even more preferably 85% or 90%, and most preferably 95% identical to SEQ ID NO:1.
By a xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and other naturally-occurring organic molecules with which it is naturally associated. Preferably, the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight. A substantially pure APP-2 polypeptide may be obtained, for example, by extraction from a natural source (e.g., mammalian platelets); by expression of a recombinant nucleic acid encoding an APP-2 polypeptide; in cells or in a cell-free system; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography such as immunoaffinity chromatography using Mab 3B2, polyacrylamide gel electrophoresis, or HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is either chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.
In addition to substantially full-length polypeptides, the invention also includes fragments of these polypeptides. As used herein, xe2x80x9cfragment,xe2x80x9d as applied to a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 60 residues in length. Fragments of the APP-2 polypeptide can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a characteristic of APP-2 (e.g., binding to Mab 3B2 or any other anti-APP-2 antibody) can be assessed by those methods described herein. Also included in the invention are APP-2 polypeptides which are encoded by portions of SEQ ID NO:2, e.g., products of alternative mRNA splicing or alternative protein processing events, or in which a section of APP-2 sequence, such as the transmembrane domain and/or intracellular domain, has been deleted. The sequence of the transmembrane domain is routinely determined by identifying a segment consisting of predominantly hydrophobic residues characteristic of a transmembrane domain.
The invention also includes a polypeptide which includes at least 20 amino acids of APP-2. Preferably, the polypeptide includes at least 50, more preferably at least 100, more preferably at least 200, and most preferably at least 300 amino acids of APP-2. Preferably, the polypeptide is an antigenic fragment of APP-2 or a soluble fragment of APP-2 which lacks the transmembrane domain of APP-2.
APP-2 has been found in covalent association with a complex (APCOM) that migrates at an apparent molecular weight of 145 kDa under non-reducing conditions. This complex contains at least two proteins in addition to APP-2: one of approximately 45 kD and one of approximately 15 kD. These proteins and the APCOM are all within the invention. By virtue of its characteristic association with activated platelets, as opposed to unactivated platelets, the APCOM and its constituent proteins can be used to generate antibodies (such as MAb 3B2) diagnostic for activated platelets and for thrombus.
The invention also includes a polyclonal or monoclonal antibody which specifically binds to the platelet activation polypeptide of the invention. Preferably, the antibody is MAb 3B2 or binds to the same epitope as MAb 3B2. The invention encompasses not only an intact monoclonal antibody, but also an immunologically-active antibody fragment, e.g., a Fab or (Fab)2 fragment; an engineered single chain Fv molecule; or a chimeric molecule, e.g., an antibody which contains the binding specificity of one antibody, e.g., of murine origin, and the remaining portions of another antibody, e.g., of human origin. In preferred embodiments, the antibody may be linked to a detectable label, e.g. a radioactive label, fluorescent label, paramagnetic label, or colorimetric label.
Also within the invention is a method of detecting an activated platelet in a biological sample, which includes the steps of contacting the sample with the labelled antibody, e.g., radioactively tagged MAb 3B2, and determining whether the antibody binds to a component of the sample. Antibody binding indicates that the sample contains an APP-2 polypeptide, and consequently, an activated platelet.
The labelled antibody may also be used diagnostically. For example, one can localize a platelet thrombus in an animal, e.g., a human patient suspected of having undesirable blood clots, by administering to the animal the labelled antibody, e.g., MAb 3B2, and determining where in the animal the label localizes. Detection of the label at a given site in the animal indicates the existence of a platelet thrombus at that site.
The antibody of the invention may also be used therapeutically, e.g., in a method of targeting a compound to an activated platelet in an animal, which includes the steps of administering to an animal a composition containing the compound linked to an anti-APP-2 antibody, e.g., MAb 3B2. Preferably, the compound is a thrombolytic agent such as urokinase, prourokinase, streptokinase, tissue-type plasminogen activator, staphylokinase, or vampire bat tissue plasminogen activator, to dissolve thrombi; an anti-thrombotic agent such as heparin, hirudin, or inhibitors of Factor Xa or Factor 5a, to inhibit thrombi formation; an anti-proliferative agent such as inhibitors of platelet-derived growth factor or heparin binding growth factor to inhibit cell proliferation, e.g., smooth muscle cell proliferation, at a thrombus site; or an anti-migration agent such as inhibitors of smooth muscle cell migration, e.g., an antibody or other specific inhibitor of urokinase or integrin function, to prevent or inhibit migration of cells which contribute to the obstruction of a blood vessel at a thrombus site.
The therapeutic agents may be linked to an anti-APP-2 MAb, e.g., MAb 3B2, using a covalent bond, such as a disulfide bond or a covalent crosslinking agent. The MAb and therapeutic agent may also be produced recombinantly, with the two components of the compound joined by a peptide bond.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.